Difference between revisions of "Part:BBa K1959001"

Revision as of 09:13, 19 October 2016

Modification of phytoene desaturase (PDS/CrtI)

The BBa_K1959002 contains the coding sequence (CDS) of phytoene desaturase (PDS), which is codon-optimized for rice (Oryza sativa) according to the CrtI sequence of Erwinia uredovora in original part: BBa_K118003 and included a Pea transit peptide of RUBISCO small subunit in its N-terminus. PDS/CrtI (EC 1.3.99.31) catalyzes the conversion of 15-cis-phytoene to all-trans-lycopene.

Usage and Biology

　Phytoene desaturase (PDS, EC 1.3.99.31) is an enzyme involved in β-carotene production. β-carotene, one intermediate product of astaxanthin biosynthesis, is converted from all-trans- lycopene by endogenous gene β-LYC in plants. Synthesis of plant lycopene is a four-step reaction regulated by four genes. While in bacteria, Erwinia uredovora, forming of lycopene is a one-step reaction catalyzed by CrtI (Figure. 1). Therefore, we used CrtI gene of Erwinia uredovora to simplify the reconstructed biosynthetic pathway of astaxanthin in our project. CrtI gene had been used and developed to standard Biobrick (BBa_K118003) by previous iGEM team. However, original CrtI gene is not suitable to express in plants. Thus, codon optimization on CrtI is made for rice. In addition, the Pea transit peptide of Rubisco small subunit was fused with the optimized CDS of CrtI, allowing plastid-import of CrtI, which makes it stable and functional. With the help of endogenous gene β-LCY, the β-carotene was synthezed in rice endosperm, stepping forward to astaxanthin biosynthesis.

Figure.1 Phytoene desaturation in plants (left) and bacteria (right).
Synthesis of lycopene in plants is a four-step reaction regulated by four genes, consisting of the two desaturases, phytoene desaturase (PDS) and f-carotene desaturase (ZDS). While in Erwinia uredovora, forming of lycopene is a one-step reaction encompassing all four desaturation steps and one cis-trans isomerization step catalyzed by CrtI.

Codon optimization of BBa_K118003

　BBa_K118003 is the coding sequence of CrtI from Erwinia uredovora, which may be unstable in rice. In order to enhance its expression efficiency, we optimized EuCrtI’s codon according to the codon bias of rice. Figure 2 shows the sequence difference between EuCrtI (BBa_K118003) and OsCrtI (for rice) (BBa_K1959002). Figure 3 shows the same amino acid sequence of EuCrtI and OsCrtI.

Figure 2 Sequence difference between EuCrtI <(BBa_K118003) and OsCrtI (BBa_K1959002).

Figure 3 Amino acid sequence of translated DNA of EuCrtI (BBa_K118003) and OsCrtI (BBa_K1959002).
Sequence alignment shows that there are no differences between amino acid sequence of original CtrI and codon-optimized CrtI.

Transcriptional Level of CrtI

　Semi-quantitative RT-PCR was performed to detect the expression level of CrtI involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and cDNA was synthesized from 1μg DNase-treated RNA.

Figure. 4 RT-PCR analyses of expression levels of CrtI gene in several transgenic rice.
Rice OsActin1 was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).

　Expected bands of the CrtI gene were observed on the gel, indicated that CrtI gene was transcribed in endosperm.

aSTRice Phenotype

　CrtI is the key enzyme of astaxanthin biosynthesis. Rice without CrtI fails to accumulate astaxanthin, appearing white in “wild type”. aSTARice contains astaxanthin and appears orange-red-color because of the coordinated expression of CrtI gene and other key astaxanthin biosynthetic genes (Figure.5). Therefore, the phenotype of aSTARice indicated that the CrtI gene is a functional gene in rice.

Figure.5 The polished rice phenotype of aSTARice.

HPLC analysis

　To further confirm the synthetic astaxanthin in aSTARice, HPLC was performed to analyze the pigment composition. Astaxanthin is identified on the basis of retention times related to standard sample. According to the retention time of standard astaxanthin sample, astaxanthin compound of extracts from transgenic rice can be confirmed (Figure.6). In addition, astaxanthin possessed the biggest peak area in the carotenoids profile, indicated that astaxanthin was the predominant carotenoid in aSTARice.

Figure 6 HPLC chromatogram of methanol extracts from transgenic aSTARice (red line) and wild-type (blue line) rice seeds. HPLC analysis recorded at 480 nm of extracts.

Sequence and Features